Hypoxic-ischemic encephalopathy caused by birth asphyxia is a leading cause of brain injury in newborns. Although neonates and adults have common responses to cerebral ischemia-hypoxia, the immature brains may also have unique pathologic mechanism that deserves special consideration for designing effective therapies. Our preliminary results indicated that ischemia-hypoxia produces a rapid induction of tissue-type and urokinase-type plasminogen activators (tPA and uPA) that coincides with transient thrombosis. However, the tPA activity persists after recovery of cerebral perfusion, and is located outside blood vessels in the ischemia/hypoxia-challenged brains. Anti-plasmin treatment reduces the extent of ischemic- hypoxic brain injury, whereas the injection of exogenous tPA worsens it. Based on these results, we hypothesize that neonates have a unique response to cerebral ischemia-hypoxia in plasminogen activator induction, which may limit thrombosis-based ischemic injury, while imposing increased risk of tPA and plasmin-mediated neurotoxicity. The present project will test this hypothesis in two specific aims. In Aim 1, we will use transgenic tPA/lacZ mice to identify the tPA-producing cells in neonatal cerebral ischemia-hypoxia. We will also use engineered mutant mice to test the roles of tPA and uPA in spontaneous fibrinolysis in this setting. In Aim 2, we will compare the effects of cerebroventricular injection of a2-antiplasmin (a Plasmin inhibitor), PAI-1 (inhibiting both tPA and uPA), and neuroserpin (tPA-specific but covering plasmin-independent toxicity of tPA) in protecting against ischemic-hypoxic brain injury in the neonates. The efficacy of these treatments will be examined at multiple time-points using different methods. In summary, the present project will test a novel mechanism of neonatal hypoxic- ischemic encephalopathy using a rodent model. Success of this project will suggest new therapy of this devastating disease in newborns. PUBLIC HEALTH RELEVANCE Hypoxic-ischemic encephalopathy caused by birth asphyxia is the single most important cause of brain injury in newborns, but its underlying mechanisms remain unclear. The central hypothesis of this proposal states that ischemia-hypoxia in the newborn brain induces acute and persistent activity of the endogenous plasminogen activators, including tPA and uPA, leading to proteolysis-type brain injury. This hypothesis will be tested in tPA-null animals and by administration of anti-tPA/Plasmin agents after neonatal cerebral ischemia-hypoxia.